1. Field of the Invention
The present invention is directed to in situ hybridization methods using nucleic acid probes for single copy sequences for detecting chromosomal structural abnormalities in fixed tissue obtained from a patient suspected of having a chromosomal structural abnormality. The probes can be labeled with either a radioactive or non-radioactive label.
2. Background of the Invention
On a genetic level, cancer is the result of the accumulation of multiple genetic changes on a cells DNA. Each alteration, whether an initiating or a progression-associated event, may be mediated through a gross chromosomal change and therefore has the potential to be cytogenetically visible. The common tumor chromosome aberrations are generally classified as structural or numerical. Structural alterations include translocations, inversions, deletions, insertions and amplifications, whereas numerical abnormalities are losses or duplications of whole chromosomes. Tumors analyzed for chromosome aberrations are broadly classified by cytogeneticists as hematological, which include leukemias and lymphomas or solid which include carcinomas and sarcomas.
Two classes of genes are implicated in cancer. Some cellular genes (the proto-oncogenes) can be activated by dominant mutations. A proto-oncogene can be converted from a normal cellular gene to an oncogene by a variety of submicroscopic events including point mutations, small insertions and deletions and juxtaposition to other chromosome sequences. This last event can be visualized cytogenetically as a translocation or inversion.
The second type of tumor genes, often referred to as tumor suppressor genes, has been isolated to date only from solid tumors. Like oncogenes, these are also normal cellular genes. However, tumor suppressor genes contribute to oncogenicity through their loss rather than through their activation, and both copies must be inactivated for tumor formation to occur. Again, there are a variety of submicroscopic mutational mechanisms by which this can occur. These are detectable at the DNA level as loss of constitutional heterozygosity in tumor DNA. Loss of the entire gene, the region of the chromosome, or even the entire chromosome will also achieve this end, and in the case of a tumor suppressor gene these chromosome deletions and losses may be detected cytogenetically.
The prognosis of malignant or premalignant lesions is in many cases correlated with the quantitative and structural aberrations in the genomic content of the disease. For example, the N-myc gene, while not a classic proto-oncogene in that it does not have a homolog carried by an acutely transforming retrovirus, is grouped with the proto-oncogenes, because of its homology with C-myc. The N-myc gene was first identified in human neuroblastoma cell lines where homogeneously staining regions (HSR) on chromosomes or double minute (DM) chromosomes were frequent. In these cell lines there is a 25 to 700-fold amplification of the N-myc gene. Amplification and/or increased expression of this gene has been found in untreated primary human neuroblastomas, retinoblastomas, glioblastomas, leukemias and carcinomas, such as small cell carcinoma of the lung. Amplification of the gene in primary neuroblastomas was found to correlate strongly with rapid disease progression and poor clinical prognosis, independent of disease stage at diagnosis. Thus, amplification of the N-myc gene appeared to be more prognostic than clinical staging of the disease.
ERBB2 (Her-2/neu) oncogene, which codes for a 185 kDa transmembrane growth factor receptor, is amplified and/or expressed in 15%-25% of breast carcinomas. Association of ERBB2 amplification and over-expression with rapid proliferation, low estrogen receptor content, and high grade of ductal carcinomas suggests that this oncogene plays an important role in the progression of breast cancer. Therefore, techniques such as flow cytometry (FCM), karyotyping and molecular techniques have been developed for the detection and characterization of such genetic changes, which may be central to the initiation and progression of neoplasms.
Flow cytometric (FCM) and morphometric analyses of cells isolated from fresh tumors or nuclei isolated from paraffin blocks have become methods for a rapid and objective screening of malignant tumors to determine the DNA index of the cells. An increase in the DNA index of certain malignancies is regarded as a prognostic parameter. Although these techniques allow the estimation of the total DNA content of large cell populations, no information about specific chromosome aberrations can be obtained and the technique cannot detect minor quantitative DNA changes.
Karyotyping of tumor cells on the other hand has been described as a more objective approach allowing a more precise determination of numerical and/or structural chromosome defects. Chromosome analysis of cancer cells by karyotyping (metaphase cytogenetics) facilitates the identification of small deviations in chromosome content and chromosome structure. However, chromosome analysis of solid cancers is in general only possible after the previous culturing of the isolated tumor cells which may result in a selective growth of cells with the highest mitotic index and loss of chromosome material. Growth of biopsy tissue under tissue culture conditions is not advantageous because the tumor contains some normal cells and it has been observed that normal cells grow faster in such conditions. Thus one cannot make an accurate determination of the percentage of tumor cells to normal cells in the biopsy. Furthermore, such analyses are often hampered by the small number of recognizable metaphases, the lack of chromosome spreading, poor banding quality, and a condensed or fuzzy appearance of the chromosomes.
The multiple molecular techniques, such as DNA sequencing, Southern and Northern Blotting, RFLP analysis and PCR make it possible to study genes, their copy number, structure and the regulation of their expression. These techniques have identified different genes involved in cancers, like proto-oncogenes and tumor suppressor genes. Although the sensitivity of these molecular techniques is high, partially as a result of the large amount of starting material, no information is obtained at the single cell level, and heterogeneity within a tumor is often difficult to detect.
It is desirable in the diagnosis of cancer to maintain the structure of the tissue taken in the biopsy so as to be able to clearly identify the tumor cells from the normal cells. In particular, it would be advantageous to observe the rare tumor cell which has invaded the normal tissue in the biopsy. However, this is not possible with current methods and accordingly, determination of the penetration of the tumor into the normal tissue for diagnostic purposes is not possible.
In situ hybridization (ISH) has been developed to overcome the limitations of FCM, karyotyping and molecular genetics. The term "interphase cytogenetics" refers to the cytogenetic analysis by means of ISH applied to non-mitotic cells. The use of chromosome specific repetitive DNA probes in combination with the ISH technique enables the detection of numerical and large structural chromosome aberrations in both metaphase spreads and interphase nuclei. However, such repetitive DNA probes are not specific for the particular gene that is interrupted or altered by the deletions, translocations, inversions or amplifications. Thus detection of the chromosomal abnormality may not be possible if the repetitive DNA sequence being detected is not included in the chromosomal structural abnormality. Thus micro-abnormalities may not be detected.
Furthermore, these studies are routinely performed on freshly isolated tumor cells or cultured cells, rather than fixed paraffinated tissues. To date, previous hybridization studies have been used on cell lines, disassociated fresh tissue or frozen tissue sections. However, typically in the clinical setting it is not always possible to work on biopsy tissue as soon as it is available, since frequently, the only tumor tissue available is paraffin embedded tissue. Furthermore the use of fixed paraffinated issue is advantageous because the tissue structure is preserved. It was believed that in situ hybridization of fixed tissue could only be done with probes for repetitive DNA sequences because the fixation prevents detection of a single copy sequence. Thus single deletions of DNA sequences could not be detected.
It is evident that methods for detecting chromosomal structural abnormalities in cells in fixed tissue using single copy probes would be advantageous.